Signaling cascades direct information. Specific and strict temporal and spatial control is essential for the fidelity of this process, as derailed signling cascades lead to disease. Here, we investigate the control of signaling cascades in neurons. If neuronal signaling goes awry, the most prominent results are disease, such as Alzheimer's disease and Down syndrome. In this proposal, we specifically focus on the role of protein ser/thr phosphatases (PSPs) in the regulation of these signaling cascades, a histrionically understudied group of proteins that control essential biological functions. In particular, we focus on Calcineurin (CN) and Protein Phosphatase 1 (PP1), which together are responsible for dephosphorylating, with high fidelity and accuracy, more than 50% of all ser/thr residues in humans. Nevertheless, a detailed molecular understanding of how this fidelity and accuracy is achieved is largely missing. This is due, in part, to the fact that this group of proteins is exceedingly difficult to work with, which has frustrated the research community for the last 25 years. The combined laboratories that are part of this application have made significant progress in understanding the structural basis of PSP function and regulation by unraveling novel mechanisms for substrate specificity and signaling pathway fidelity. Our aims are: (1) to gain fundamental, molecular insights into CN substrate recognition, which, compared to our knowledge of substrate recognition by kinases, lags decades behind; (2) to elucidate the molecular mechanism by which CN is potently regulated by RCAN1, a trisomy 21 protein that leads to dysregulation of NFAT signaling and hyper-phosphorylation of tau, leading to Down syndrome and Alzheimer's disease, respectively; and (3) to establish how PP1 regulates translation initiation, setting the stage for the identification of novel routes for treating proten misfolding diseases such as Alzheimer's. As demonstrated over the last 10 years, we use our PSP: protein holoenzyme structures to develop function models, which are then tested in vivo in cells and neurons in collaboration with the Nairn Laboratory at Yale University, the Cyert Laboratory at Stanford University and the Shenolikar Laboratory at Duke-NUS. Thus, we integrate our structural and functional studies in order to obtain the comprehensive understanding necessary to fully explain the underlying biology of PSPs, ultimately identifying novel routes for the treatment of a variety of neurological disorders. Overall, this proposal rests on an outstanding foundation of: (1) 30 years of functional data that is waiting for a molecular interpretation to provide a deeper understanding and analysis of these critical PSP holoenzymes, (2) specific, highly technical skill sets for working with PP1 and CN, (3) strong preliminary results, including the first PSP: substrate model and (4) an outstanding assembled team that has successfully collaborated for more than 10 years to elucidate the molecular basis of PSP regulation. Therefore, the proposed structural and functional studies will provide a detailed understanding of the roles of CN and PP1 in the brain and will enable us to selectively modulate these particular signaling cascades for medical benefit.